We study how enhancers activate transcription in the chromatin environment of eukaryotic cells. We used chromatinized, stably replicating episomes in human erythroid K562 cells to study the interaction of globin genes and elements of the beta-globin locus control region (LCR). Mutagenesis studies which systematically eliminated transcription factor binding sites in LCR HS2 and in the embryonic epsilon-globin promoter, revealed that enhancer and promoter mutually affect each others chromatin structure. These data support a direct communication model of enhancer action. Furthermore, an intact TATA box was required for promoter remodeling by the enhancer. When the epsilon-globin gene is transcriptionally activated by HS2, the structure of the TATA proximal nucleosome (N1) is altered. To elucidate the nature of this alteration and the link between chromatin modification and gene expression, we examined nucleoprotein composition and histone acetylation at transcriptionally active and inactive promoters. There was a marked decrease in nucleoprotein complexes recovered from the proximal promoter region of actively transcribing epsilon-globin genes compared to inactive promoters. Furthermore, the N1 nucleosome of active promoters showed dramatic hyperacetylation of histone H3 and H4, while the adjacent upstream nucleosome was not differentially acetylated. This highly directed and specific nucleosome modification that accompanies transactivation by HS2 could not be mimicked by global acetylation of histones in vivo by Trichostatin A, suggesting additional activities of HS2 are necessary for transactivation. Taken together, the data argue that N1 is present but in an altered conformation in at least some of the active promoters. We continue to explore the regulatory role in vivo of chromatin structure in the expression of globin genes, and the mechanism of action of the beta-globin LCR. - globin genes, chromatin structure, transcription, locus control region, nucleosomes, histone acetylation enhancers"